A wide variety of chemical moieties (‘payloads’) have been covalently attached to enzymes, antibodies, and other polypeptides or proteins, to form conjugates. The payloads may be used to locate the protein to which they are attached (e.g., labels), to modify the physicochemical properties or stability of the protein (e.g., PEGylation), to enable the protein to be attached to another molecule or protein (functional or coupling groups, for connecting the conjugate to another compound or another conjugate), or to modify the function or activity of the payload or the protein (e.g., vaccine conjugates). The protein may also act as a carrier to deliver the attached payload to a particular tissue or cell type, such as in antibody-drug conjugates (ADCs). Classes of payloads that can be usefully linked to proteins include detectable moieties (labels), anchoring moieties to attach the protein to a surface or another compound, antigens that elicit an immune response when conjugated to a protein, coupling groups that react readily with a chemically complementary coupling partner (thus connecting the protein to another entity), and therapeutic moieties such as cytotoxins and other bioactive agents.
Attaching these diverse structures to proteins in a controlled and reproducible fashion is often critical for the conjugates to function correctly, particularly when they are used as therapeutic agents. In ADCs, for example, it is important to carefully control the number of payload compounds attached to an antibody, which is not easy since the carrier is a large and complex protein. For example, depending on the particular target, linker, and cytotoxin, the optimum DAR (drug to antibody ratio) for an ADC may vary from 1 to 6 or more, in order to balance factors such as efficacy, stability, and safety. If ADCs are made as mixtures with varying drug/antibody ratios (DARs), separation is difficult, and consistency of the product is important for both therapeutic and regulatory reasons. Thus there is a need for methods to produce ADCs with good control of drug-antibody ratios, as well as efficient conjugation and consistent placement of the payload attachment point.
A number of methods have been developed for attaching payloads to proteins to address some of these issues. See, e.g., Sletten, E. M. and Bertozzi, C. R. Angew. Chem. Int. Ed. 2009, 48, 6974-6998; Basle', E.; Joubert, N.; Pucheault, M. Chemistry & Biology 2010, 17, 213-227; and Hermanson, G. T. Bioconjugate Techniques, 2nd ed.; Academic Press: San Diego, Calif., 2008. The most common conjugation methods rely on the chemical reactivity of certain amino acids that occur naturally in many natural proteins: lysine and cysteine are often used, because they provide a reactive site for connecting the payload to the protein. Proteins often have more lysines than the optimum number of payloads to be attached, though: adding enough payload moieties to occupy all of the availably lysines in order to produce a consistent, homogenous product may add too many payload molecules for optimum efficacy, while partial loading typically provides a heterogeneous product, which can be problematic for a variety of reasons—in the case of Mylotarg™, the first commercialized ADC, for example, the heterogeneity of the ADC product seems likely to have contributed to the issues that led to a decision to withdraw the product from registration. Fuenmayor, et al., Cancers, vol. 3, 3370-93 (2011).
The frequency of occurrence of cysteine in natural proteins is lower than that of lysine, and cysteine may be suitable for use as a site for conjugation where it is available in adequate numbers; where too few cysteines are present, one or more may be inserted by standard protein modification methods. However, it is often preferable to avoid modifying the sequence of the natural protein by inserting a cysteine or by removing a disulfide. While it is not difficult to convert a disulfide into two free cysteines by reducing the disulfide, doing so may disrupt the secondary or tertiary structure of the protein.
Some methods for inserting a tether between cysteine residues formed by reducing a disulfide on a protein have been reported. This approach is particularly appealing for ADC production, because the typical antibody structure contains four reducible inter-chain disulfides that can be utilized without protein engineering. One method used for such conjugation involves a sulfone-substituted methacrylate derivative. US2006/0210526. This method forms a reactive intermediate that requires an elimination step before alkylation of the second sulfur atom occurs. The conditions for that multi-step process can result in incomplete formation of a linker (tether) between cysteines, and the reaction conditions can cause protein denaturation. Another approach uses a maleimide derivative, e.g., a 3,4-dibromomaleimide. WO2011/018613. However, the conjugate formed in this process can suffer from stability problems because the Michael addition of the thiols on the maleimide ring is reversible, so the tether between the sulfur atoms or the payload itself can be lost. Thus novel methods are needed that turn disulfide groups into conjugation sites without giving up the stabilizing effect of the inter-chain disulfides, while also providing efficient conjugation, stability, and consistent payload/protein ratios. The present invention provides useful methods for making such ADCs.